EP0063494A2 - Méthode de production de protéine à partir de micro-organismes, micro-organismes à utiliser pour cette méthode et leur création, vecteurs à utiliser dans cette création, protéine produite par ce micro-organisme, et culture transformée dérivée de ces micro-organismes - Google Patents

Méthode de production de protéine à partir de micro-organismes, micro-organismes à utiliser pour cette méthode et leur création, vecteurs à utiliser dans cette création, protéine produite par ce micro-organisme, et culture transformée dérivée de ces micro-organismes Download PDF

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Publication number
EP0063494A2
EP0063494A2 EP82302027A EP82302027A EP0063494A2 EP 0063494 A2 EP0063494 A2 EP 0063494A2 EP 82302027 A EP82302027 A EP 82302027A EP 82302027 A EP82302027 A EP 82302027A EP 0063494 A2 EP0063494 A2 EP 0063494A2
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EP
European Patent Office
Prior art keywords
heterologous gene
subtilis
protein
plasmid
initiation codon
Prior art date
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Granted
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EP82302027A
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German (de)
English (en)
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EP0063494A3 (en
EP0063494B1 (fr
Inventor
Shing Chang
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Novartis Vaccines and Diagnostics Inc
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Cetus Corp
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Priority to AT82302027T priority Critical patent/ATE48639T1/de
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Publication of EP0063494A3 publication Critical patent/EP0063494A3/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • This invention relates to molecular biology and, more particularly, to the so-called art of recombinant DNA.
  • the inventipn relates to a method and cloning vector for the production of cloned heterologous gene products in Bacillus subtilis as single urfused proteins.
  • mRNA messenger RNA
  • codons groups of three nucleotides, called codons, in the mRNA direct the placement of one of twenty possible amino acids at a corresponding position in the protein chain.
  • predetermined nucleotide sequence may be selected to cause the strain or species into which it is introduced to produce, as part of the translation process, the protein encoded by the predetermined nucleotide sequence.
  • modified strain or species proceeds with the normal replication process, it also then duplicates the inserted sequence.
  • Recombinant DNA techniques involve isolating a suitable piece of DNA chain (a cloning vector) and breaking or severing the two strands of DNA of the cloning vector at the desired location where the foreign D N A is to be inserted.
  • a suitable piece of DNA chain a cloning vector
  • restriction enzymes particular types of proteins, called restriction enzymes. Restriction enzymes will break the DNA at particular nucleotide sequences, although with some restriction enzymes the break may not necessarily occur at the same point on the two intertwined DNA strands.
  • the open ends will be complementary and will, under suitable conditions, stick together with the complementary ends lying side by side. They may then be linked together enzymatically with an enzyme such as a ligase. This makes it possible to recombine two DNA segments from any source into a single DNA molecule.
  • the recombinant DNA is then placed into a suitable host organism.
  • the host organism In order for the host organism to replicate the inserted DNA, it is necessary that the recombinant DNA be inserted into the host in such a way as to become part of its genetic system.
  • Such insertion can occur in a variety of ways.
  • Escherichia coli two convenient types of cloning vectors have been utilized.
  • E. coli bacteria in addition to the main DNA chain or chromosome, frequently have one or more independently replicating circular loops of DNA known as plasmids.
  • a certain type of virus known as a lambda bacteriophage (phage) is also capable of infecting E.
  • plasmids or phages as cloning vectors. This involves the isolation of plasmids or phages from the bacteria, the breaking open of the isolated DNA by restriction enzymes, the insertion of a foreign or heterologous piece of DNA into the plasmid or phage so that it is correctly orientated, the restoration of the circular form of the plasmid or the phage structure, and the return of the plasmid or phage to the E. coli cell.
  • the heterologous DNA is not only replicated from generation to generation, but also will produce the protein for which it codes if it is correctly oriented and if the proper reading frame and promoters exist.
  • E. coli is a prokaryotic cell which has, instead of a true nucleus, a single "naked" chromosome not surrounded by a nuclear membrane.
  • Yeast are eukaryotes since they contain a true nucleus.
  • Man is also a euykaryote, although one quite distinct from yeast since the two eukaryotes diverged from a common ancestor more than 1.2 billion years ago. Because of the distinct differences between man and yeast, it is not surprising that the two organisms are separated by an evolutionary gap of more than 1.2 billion years. More surprising is the fact that bacterial species can be separated by a similar evolutionary gap.
  • E. coli plasmids are not usually capable of replication in B. subtilis and E. coli genes are not generally expressed in B. subtilis. This is sometimes due to differing genetic control mechanisms that result from the great evolutionary gap between the two bacterial species. See, Hori and Osawa, supra.
  • B. subtilis is preferable to E. coli as a host organism because of a greater efficiency for plasmid mediated transformation in B. subtilis and because it is non-pathogenic.
  • B. subtitlis is also a fermentation organism used extensively in industry; therefore its management on an industrial scale is familiar.
  • the laboratory E. coli strain is a non-pathogenic organism native to the intestines of man and higher animals, there is concern that it might become disease producing if genetically altered.
  • B. subtilis on the other hand, lives in the soil and has never been associated with any disease of plants or animals. Thus there is less likelihood that a genetic alteration in B. subtilis would lead to a disease state in man or animals or plants.
  • the present invention relates to an advance in the basic technology of the aforesaid Application, in that it demonstrates the first successful production of unfused heterologous protein in B. subtilis in which only the desired unfused or pure form of the heterologous protein is produced by the bacteria.
  • the unfused or pure form of the protein product has no extraneous amino acids coded for by sources other than the heterologous gene.
  • the pure heterologous protein is allowed to accumulate within the host. When a desired amount of the pure heterologous protein has accumulated within the host, the pure protein can'be recovered by means known to the art.
  • the invention provides a method for producing a predetermined protein that will accumulate within a transformed host comprising providing growth conditions in a growth media for B. subtilis bacteria containing plasmids capable of replication in B. subtilis or capable of integrating into the bacterial chromosome of B.subtilis, said plasmids having a heterologous gene therein coding for a predetermined protein capable of expression in B .
  • said heterologous gene including a translational initiation codon sequence and being under the control of operable transcriptional and translational regulatory signals including operator, promoter and ribosomal binding site sequences, said operable transcriptional and translational regulatory signals being indigenous to a source other than said heterologous gene and further being absent any translational initiation codon sequences prior to said heterologous gene, and optionally recovering said predetermined protein.
  • the invention includes a microorganism-capable of producing a predetermined protein through expression, comprising a B. subtilis host microorganism and a plasmid capable of replication in B. subtilis or capable of integrating into the B. subtilis bacterial chromosome, said plasmid having a heterologous gene therein coding for a predetermined protein capable of expression in B. subtilis, said heterologous gene including a translational initiation codon sequence and being under the control of operable transcriptional and translational regulatory signals including operator, promoter and ribosomal binding site sequences, said operable transcriptional and translational regulatory signals being indigenous to a source other than said heterologous gene and further being absent any translational initiation codon prior to said heterologous gene.
  • a method for creating a microorganism capable of producing a predetermined protein through expression comprising forming a plasmid capable of replication in B. subtilis or capable of integrating into the B. subtilis bacterial chromosome, and having a heterologous gene therein coding for a predetermined protein capable of expression in B.
  • subtilis said heterologous gene including a translational initiation codon sequence and being under the control of operable transcriptional and translational regulatory signals including operator, promoter and ribosomal binding site sequences, said operable transcriptional and translational regulatory signals being indigenous to a source other than said heterologous gene and further being absent any translational initiation codon sequences prior to said heterologous gene, and introducing said plasmid to B. subtilis.
  • the invention naturally also provides a plasmid capable of replication in B. subtilis or capable of integrating into the B. subtilis bacterial chromosome, said plasmid having a heterologous gene therein coding for a predetermined protein capable of expression in B. subtilis, said heterologous gene including a translational initiation codon sequence and being under the control of operable transcriptional and translational regulatory signals including operator, promoter and ribosomal binding site sequences, said operable transcriptional and translational regulatory signals being indigenous to a source other than said heterologous gene and further being absent any translational initiation codon prior to said heterologous gene.
  • a predetermined single unfused, protein which is non-indigenous or heterologous to B. subtilis is produced through expression by B. subtilis.
  • Growth media and conditions are provided for growing a strain of B . subtilis in which a plasmid has been introduced.
  • the plasmid is capable of being replicated in the strain or is capable of being integrated into the bacterial chromosome.
  • Such a plasmid carries a properly oriented heterologous gene that codes for the desired predetermined protein.
  • the heterologous gene contains its own translational initiation codon sequence. This translational initiation codon can be naturally present on the heterologous gene or can be placed there synthetically.
  • Appropriate transcriptional and translational regulatory signals including operator, promoter and ribosomal binding site sequences, from a source other than the heterologous gene, are also present in the plasmid.
  • the heterologous gene containing its own translational initiation codon sequence, is located on the plasmid behind a ribosomal binding site sequence at a distance sufficient to provide the spacer nucleotides necessary for proper translation of the heterologous gene. There are no translational initiation codon sequences on the plasmid between the regulatory signals and the point at which the heterologous gene is located.
  • heterologous gene is under the control of plasmid regulatory signals, the presence of the translational initiation codon sequence on the heterologous gene insures that the desired initiation of translation will begin at the site of the heterologous gene. As a result, the heterologous gene is expressed as a single unfused peptide.
  • the heterologous gene product will accumulate in the host organism as an intracellular protein. The heterologous gene product can be recovered from within the host organism by means known in the art.
  • the human fibroblast interferon gene for use in the expression of the human fibroblast interferon in B. subtilis was isolated by means known to the art. See Taniguchi et al, Proc.Jpn.Acad., Ser.B 55:464-469 (1979). More specifically, the human fibroblast interferon cDNA clone, 4El,was obtained by reverse-transcriptase synthesis of cDNA using human mRNA as template and oligo-dT as primer.
  • the cDNA was made double stranded by the action of E.coli DNA polymerase I and nicked with Sl-nuclease.Homopolymeric tails were added to the 3'-terminal of the double stranded cDNA (dscDNA)by the enzyme terminal-transferase using dCTP as substrate. Similar dG homopolymeric tails were added to the 3'-termini of the plasmid pBR322 which had been linearized at the PstI site. Plasmid pBR322 is on deposit with the American type culture collection. It has been awarded ATOC number 37,017. Sanples of the deposit are available to anyone without restriction. The vector and the dscDNA were hybridized and transformed into E.
  • the clone, 4El was identified by Grunstein-Hogness colony hybridization screens using a p 32- labeled probe and further characterized by restriction enzyme analysis. Such analysis showed that PstI digestion of 4El yields two insert fragments of about 600 bp and 200 bp, in addition to a fragment corresponding to linear p B R322. BglII and PstI digestion of the same clone showed that the 600 PstI insert fragment can be further digested with BglII to yield two fragments of sizes 358 bp and 200 bp. HinfI digestion of clone 4El showed that there are at least three HinfI sites in the insert fragment which generates the three new fragments now present in pBR322.
  • the human fibroblast interferon gene Prior to its expression in B. subtilis, the human fibroblast interferon gene was subcloned for expression in E. coli. To accomplish this, the human fibroblast interferon gene coding sequence was subcloned by using a synthetic oligonucleotide primer (TATGAGCTACAAC) and the enzyme DNA polymerase I to degrade DNA sequences 5' to the ATG codon that codes for the amino-terminal methionine of the mature interferon. The repaired.DNA was then subcloned into pBR322 at the repaired HindIII and at the BamHl sites.
  • TATGAGCTACAAC synthetic oligonucleotide primer
  • the BglII site in the human interferon gene just past the UGA translation termination codon, was used to ligate with the BamHl cohesive end in pBR322 to regenerate an XhoII site.
  • the resulting clone pl-25 was confirmed by restriction analysis and DNA sequence analysis.
  • a bacillus penicillinase (beta-lactamase) promoter was used to express the human fibroblast interferon in B. subtilis.
  • the bacillus penicillinase (beta-lactamase) promoter fragment was generated from a cloned penicillinase gene. See Gray and Chang, J. B acteriol.145:422-428 (1981).
  • the coding sequence of the penicillinase (penP) gene was digested at the PstI site. Then the linearized DNA was trimmed with BAL-31 exonuclease to remove the coding region just beyond the A TG codon.
  • the coding sequence is outlined in FIGURE 2.
  • the DNA was then further digested at the EcoRl'site located at the 5' end, and repaired by DNA polymerase to generate a blunt-ended fragment. This fragment was further fractionated and purified on acrylamide gel.
  • the purified DNA containing the penP gene promoter was then cloned into pLL10 at the Smal site by blunt-end ligation. See Wu, R., (Editor) Methods in Enzymology, 68:98-109 (1979). Since the SmaI site is flanked by two B amHl sites, it was possible to excise the promoter fragment by BamHl digestion.
  • the E. coli beta-1-25 plasmid was first converted to a bifunctional replicon by in vitro ligation with bacillus plasmid pOG1196 at. the PvuII site.
  • the BamHl fragment containing the 262 bp penP promoter was then cloned into the HindIII site 5' to the human fibroblast interferon coding sequence following limited Sl nuclease digestion and E. coli DNA polymerase repair.
  • One resulting clone, pDH1151 was further characterized by restriction enzyme analysis. Such analysis showed that in addition to the Bacillus derived sequences, this plasmid carried only sequences derived from E.
  • Plasmid pDH1151 was then transformed into the B. subtilis HV1 host strain BGSClS53. Cell extract prepared from the transformed strain carrying pDH1151 showed that the resulting strain produces intracellular human fibroblast interferon exhibiting antiviral activity comparable to that shown by single unfused mature human fibroblast interferon. See Stewart, in The Interferon System, Springer-Verlag, New York (1979) at pp. 17-18 for details of antiviral assay.
  • the invention provides a method and a vector for the expression of heterologous cloned genes as single unfused proteins in B. subtilis. These unfused heterologous proteins will not contain any extraneous amino acids coded for by DNA originating from either the host or the plasmid. These single unfused protein products will accumulate within the host organism. Following their accumulation within the host organism; the heterologous gene products can be recovered by means known in the art.
  • Plasmid pOG1196 referred to hereinabove is described in Gray & Chang, S., J. Bacteriol. 145:422-428 (1981).
  • Figure 4 gives the map for this plasmid.
  • the plasmid has been deposited with the American type culture collection, Rockville, Maryland 02852, U.S.A.
  • the deposited plasmid has been awarded ATCC number 31,776.

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EP82302027A 1981-04-20 1982-04-20 Méthode de production de protéine à partir de micro-organismes, micro-organismes à utiliser pour cette méthode et leur création, vecteurs à utiliser dans cette création, protéine produite par ce micro-organisme, et culture transformée dérivée de ces micro-organismes Expired EP0063494B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT82302027T ATE48639T1 (de) 1981-04-20 1982-04-20 Verfahren zum produzieren von protein aus einem mikroorganismus, mikroorganismen zur verwendung in solchem verfahren und ihre erzeugung, vektoren zur verwendung in dieser erzeugung und protein produziert durch diesen mikroorganismus und von diesen mikroorganismen abgeleitete transformierte kulturen.

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Application Number Priority Date Filing Date Title
US25580481A 1981-04-20 1981-04-20
US255804 1981-04-20

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EP0063494A2 true EP0063494A2 (fr) 1982-10-27
EP0063494A3 EP0063494A3 (en) 1984-02-22
EP0063494B1 EP0063494B1 (fr) 1989-12-13

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EP (1) EP0063494B1 (fr)
JP (1) JPS58500590A (fr)
AT (1) ATE48639T1 (fr)
AU (1) AU551529B2 (fr)
CA (1) CA1204681A (fr)
DE (1) DE3280060D1 (fr)
DK (1) DK563082A (fr)
ES (2) ES8308586A1 (fr)
FI (1) FI824238A0 (fr)
IL (1) IL65558A0 (fr)
NO (1) NO824102L (fr)
WO (1) WO1982003633A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0063953A2 (fr) * 1981-04-29 1982-11-03 Biogen N.V. Vecteurs de clonage de bacillus, molécules de DNA recombinant, hôtes de bacillus transformés par celles-ci et méthode pour l'expression de séquences de DNA étrangères et pour la production de polypeptides codés par ce DNA
EP0116411A2 (fr) * 1983-01-18 1984-08-22 Eli Lilly And Company Vecteurs d'expression ADN utilisables dans des cellules de Bacillus
EP0117041A2 (fr) * 1983-01-18 1984-08-29 Eli Lilly And Company Séquence ADN dans des streptomyces
US4585739A (en) * 1983-03-07 1986-04-29 E. I. Du Pont De Nemours And Company Plasmid for foreign gene expression in B. subtilis
US4595660A (en) * 1983-03-02 1986-06-17 University Of Delaware Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis, mutants and substantially stably transformed mutants of Bacillus subtilis, and methods for utilizing the transformed mutants
US4663280A (en) * 1983-05-19 1987-05-05 Public Health Research Institute Of The City Of New York Expression and secretion vectors and method of constructing vectors
US4705750A (en) * 1983-12-26 1987-11-10 Takeda Chemical Industries, Ltd. Promoter plasmid containing the promoter and use thereof in transforming Bacillus
US4769327A (en) * 1985-03-29 1988-09-06 Biotechnica International, Inc. Secretion vector
US5310675A (en) * 1983-06-24 1994-05-10 Genencor, Inc. Procaryotic carbonyl hydrolases

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1186750B (it) * 1985-07-10 1987-12-16 Eniricerche Spa Vettore di clonaggio,molecole di dna ricombinante,ceppi di bacillus subtilis trasformati con dette molecole e metodi per l'espressione di geni eterologhi e produzione e secrezione di proteine codificate da detti geni

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2039916A (en) * 1979-01-15 1980-08-20 Harvard College Protein synthesis by genetic manipulation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0005476B1 (fr) * 1978-05-17 1981-12-30 Dr. Karl Thomae GmbH Procédé de préparation d'interféron humain
JPS5564799A (en) * 1978-11-07 1980-05-15 Toray Ind Inc Multi-stage concentration and purification of interferon originated from human fibroblast
US4311639A (en) * 1980-07-25 1982-01-19 E. I. Du Pont De Nemours And Company Immunogenic interferon peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2039916A (en) * 1979-01-15 1980-08-20 Harvard College Protein synthesis by genetic manipulation

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BACTERIOLOGY, vol. 145, no. 1, January 1981, pages 422-428 *
NATURE *
NATURE, vol. 281, 18th October 1979, pages 544-548, Macmillan Journals Ltd. *
NATURE, vol. 287, 02 Oct 1980, Macmillan Journals Ltd.; D.V.GOEDDEL et al.: " Human leucocyte interferon produced by E.coli is biologically active", pp. 411-416 *
NATURE, vol. 287, 2nd October 1980, pages 411-416, Macmillan Journals Ltd. *
PROC. NATL. ACAD. SCI. USA, vol. 77, no. 9, September 1980, pages 5230-5233 *
SCIENCE, vol. 209, 19th September 1980, pages 1428-1430, AAAS *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0063953A2 (fr) * 1981-04-29 1982-11-03 Biogen N.V. Vecteurs de clonage de bacillus, molécules de DNA recombinant, hôtes de bacillus transformés par celles-ci et méthode pour l'expression de séquences de DNA étrangères et pour la production de polypeptides codés par ce DNA
EP0063953A3 (fr) * 1981-04-29 1983-10-19 Biogen N.V. Vecteurs de clonage de bacillus, molécules de DNA recombinant, hôtes de bacillus transformés par celles-ci et méthode pour l'expression de séquences de DNA étrangères et pour la production de polypeptides codés par ce DNA
EP0116411A2 (fr) * 1983-01-18 1984-08-22 Eli Lilly And Company Vecteurs d'expression ADN utilisables dans des cellules de Bacillus
EP0117041A2 (fr) * 1983-01-18 1984-08-29 Eli Lilly And Company Séquence ADN dans des streptomyces
EP0117041A3 (en) * 1983-01-18 1986-03-05 Eli Lilly And Company Dna sequence in streptomyces
EP0116411A3 (en) * 1983-01-18 1986-03-19 Eli Lilly And Company Dna expression vectors useful in bacillus cells
US4595660A (en) * 1983-03-02 1986-06-17 University Of Delaware Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis, mutants and substantially stably transformed mutants of Bacillus subtilis, and methods for utilizing the transformed mutants
US4585739A (en) * 1983-03-07 1986-04-29 E. I. Du Pont De Nemours And Company Plasmid for foreign gene expression in B. subtilis
US4663280A (en) * 1983-05-19 1987-05-05 Public Health Research Institute Of The City Of New York Expression and secretion vectors and method of constructing vectors
US5310675A (en) * 1983-06-24 1994-05-10 Genencor, Inc. Procaryotic carbonyl hydrolases
US4705750A (en) * 1983-12-26 1987-11-10 Takeda Chemical Industries, Ltd. Promoter plasmid containing the promoter and use thereof in transforming Bacillus
US4769327A (en) * 1985-03-29 1988-09-06 Biotechnica International, Inc. Secretion vector

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ATE48639T1 (de) 1989-12-15
NO824102L (no) 1982-12-07
ES511510A0 (es) 1983-09-16
ES8403158A1 (es) 1984-03-01
AU551529B2 (en) 1986-05-01
CA1204681A (fr) 1986-05-20
JPS58500590A (ja) 1983-04-21
ES8308586A1 (es) 1983-09-16
AU8521382A (en) 1982-11-04
EP0063494A3 (en) 1984-02-22
WO1982003633A1 (fr) 1982-10-28
IL65558A0 (en) 1982-07-30
EP0063494B1 (fr) 1989-12-13
FI824238L (fi) 1982-12-09
FI824238A0 (fi) 1982-12-09
ES519234A0 (es) 1984-03-01
DE3280060D1 (de) 1990-01-18
DK563082A (da) 1982-12-20

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